Generation of mesenchymal stromal cells from urine-derived iPSCs of pediatric brain tumor patients

Abstract

Human induced pluripotent stem cells (iPSCs) provide a virtually inexhaustible source of starting material for next generation cell therapies, offering new opportunities for regenerative medicine. Among different cell sources for the generation of iPSCs, urine cells are clinically relevant since these cells can be repeatedly obtained by non-invasive methods from patients of any age and health condition. These attributes encourage patients to participate in preclinical and clinical research. In particular, the use of urine-derived iPSC products is a convenient strategy for children with brain tumors, which are medically fragile patients. Here, we investigate the feasibility of using urine samples as a source of somatic cells to generate iPSC lines from pediatric patients with brain tumors (BT-iPSC). Urinary epithelial cells were isolated and reprogrammed using non-integrative Sendai virus vectors harboring the Yamanaka factors KLF4, OCT3/4, SOX2 and C-MYC. After reprogramming, BT-iPSC lines were subject to quality assessment and were compared to iPSCs obtained from urine samples of non-tumor pediatric patients (nonT-iPSC). We demonstrated that iPSCs can be successfully derived from a small volume of urine obtained from pediatric patients. Importantly, we showed that BT-iPSCs are equivalent to nonT-iPSCs in terms of morphology, pluripotency, and differentiation capacity into the three germ layers. In addition, both BT-iPSCs and nonT-iPSCs efficiently differentiated into functional mesenchymal stem/stromal cells (iMSC) with immunomodulatory properties. Therefore, this study provides an attractive approach to non-invasively generate personalized iMSC products intended for the treatment of children with brain tumors.

Keywords: cancer; cell reprogramming; cell therapy; central nervous system cancer; children; iPSC; mesenchymal stem/stromal cells (MSC); oncology.

Figure 1

Representative magnetic resonance images of pediatric patients with brain tumors included in the study. Saggital and axial images reveal a low grade glioma (BT Patient 1, 43-month-old) and a metastatic brain tumor (BT Patient 2, 24-month-old). Red arrows point to the tumor.

Figure 2

Isolation of UDCs and reprogramming process. (A) Schematic timeline of the process for isolation and expansion of UDCs and iPSC generation. (B) Microscope images showing the morphological aspect of cultured UDCs (scale bar 200 µm), iPSC colonies obtained after selective passaging post-reprogramming (scale bar 1 mm), iPSCs at higher magnifications (scale bar 100 µm) and alkaline phosphatase staining of iPSC colonies (scale bar 400 µm).

Figure 3

Analysis of the pluripotency markers in the generated iPSC lines. (A) RT-PCR analysis of the expression of pluripotency-associated genes. Note that all iPSC lines highly express OCT3/4, SOX2, NANOG and TERT, while UDCs do not. (B) Immunofluorescence staining showing the presence of pluripotency markers OCT3/4 (magenta), SSEA-4 (green), TRA-1-60 (green) and TRA-1-81 (green) in the iPSCs. Nuclei were counterstained with Hoechst 33342 (blue). Scale bar 100 µm.

Figure 4

Analysis of the differentiation capacity of the generated iPSCs into the three embryonic layers. (A) RT-PCR analysis showing the expression of specific differentiation markers, including markers for endoderm (SOX17 and FOXA2), mesoderm (CXCR4 and BRACH) and ectoderm (NGN3). Note that differentiated iPSCs highly express all the trilineage differentiation markers, while undifferentiated iPSCs do not. (B) Immunofluorescence staining showing the presence of alfa-fetoprotein (AFP) for endoderm, Smooth Muscle Actin (SMA) for mesoderm and Nestin for ectoderm in the differentiated iPSCs. Nuclei were counterstained with Hoechst 33342. Scale bar 50 µm.

Figure 5

Authentication, molecular karyotyping, virus clearance and mycoplasma testing of established iPSCs. (A) DNA fingerprint analysis showing that the allele pattern in the iPSCs generated is 100% concordant with the patients’ UDCs and it is not concordant with any commercial cell line whose genotype is posted in public databases. The STR locations studied were: TH01, D21S11, D5S818, D13S317, D7S820, D16S539, CSF1PO, vWA, TPOX and AMEL. The percentage of matching between iPSCs and their parental UDCs is indicated for each sample. (B) Whole genome view of the iPSC lines which displays all somatic and sex chromosomes in one frame. The smooth signal plot (right y-axis) is the smoothing of the Log2 ratios (left y-axis), which depict the signal intensities of probes on the microarray and represents the number of copies of each chromosome. The pink, green and yellow colors represent the raw signal for each individual chromosome probe, and the blue signal represents the normalized probe signal, used to identify copy number and any aberrations. The percentage of autosomal loss of heterozygosity (LOH) is indicated for each cell line. (C) RT-PCR analysis showing the absence of expression of Sendai virus (SeV) genome and OSKM transgenes in the established iPSC lines. UDCs served as negative control and recently transfected iPSCs served as positive control. (D) PCR test for mycoplasma detection, showing the absence of contamination in cultured iPSC lines.

Figure 6

Directed differentiation of iPSCs towards iMSCs. (A) Schematic timeline of the process for differentiation into iMSCs. (B) Microscope images showing the morphological aspect of differentiated iMSCs over time (scale bar 100 µm). (C) Flow cytometry analysis of differentiated iMSCs showing that cells were positive for the MSC-specific markers CD13, CD73, CD90 and CD105, whereas they were negative for CD14, CD34, CD45, and HLA-II. (D) Representative images of the differentiated iMSCs into osteocytes (scale bar 100µm), adipocytes (scale bar 50µm) and chondrocytes (scale bar 100µm) identified by Alizarin Red, Oil Red O and Alcian Blue staining, respectively.

Figure 7

Immunomodulatory potential of the generated iMSCs. (A) Bar graph showing the fluorescence intensity for inflammatory cytokines secreted by iMSCs determined by ELISA. (B) Inhibitory effect of iMSCs on CD3+ T cell proliferation after 5 days co-culture. Dashed line indicates the % of proliferation for CD3+ T cells without iMSCs. (C) Bar graphs showing the percentage of CD3⁺, CD4⁺ and CD8⁺ proliferating T cells in response to the secretome of primed or non-primed iMSCs. Of note, the secretome was collected 48 hours after priming. (D) Bar graphs showing the expression levels of immunomodulatory genes analyzed by RT-qPCR in primed and non-primed iMSCs derived from BT patients. Of note, the RNA was isolated immediately after priming. (E) Bar graphs showing the expression levels of immunomodulatory genes analyzed by RT-qPCR in primed and non-primed iMSCs derived from BT patients. Of note, the RNA was isolated 48 hours after priming. Data are represented as mean ± SEM. The absence of bar in any graph indicates undetected levels of the specific parameters assessed. For (A); *p*0.05, ***p<0.001, ****p<0.0001 compared to nonT-iMSCs-2; ^#p<0.05, ^##p<0.01, ^####p<0.0001 compared to nonT-iMSCs-1; $$$p<0.001, $$$$p<0.0001 compared to BT-iMSCs-1. One-way ANOVA. For (B); P value refers to dose effects. Two-way ANOVA. For (C); *p<0.05, ****p<0.0001 compared to stimulated PBMC cultured in regular growth medium. ^#p<0.05, ^##p<0.01, ^###p<0.001, ^####p<0.0001 compared to their primed counterparts. One-way ANOVA. For (D-E); *p<0.05, ***p<0.001. Student's t-test.